Blackburn:Yeast Colony PCR - Revision history

Tetm2002 at 01:13, 21 March 2009

Tetm2002 — Sat, 21 Mar 2009 01:13:34 GMT

←Older revision		Revision as of 01:13, 21 March 2009
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		+	Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0\|Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0]]
		+
	==Overview==		==Overview==

Tetm2002 — Fri, 20 Mar 2009 23:00:16 GMT

Overview

←Older revision		Revision as of 23:00, 20 March 2009
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	This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.		This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.
		+
		+	Updated Protocol: [[Blackburn:Yeast_Colony_PCR_v2.0\|Blackburn Lab: Quick and Easy Yeast Colony PCR v2.0]]

	==Materials==		==Materials==

Tetm2002 — Mon, 12 Jun 2006 04:29:53 GMT

Yeast Cell Lysis

←Older revision		Revision as of 04:29, 12 June 2006
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	#Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.		#Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
	#*In the mean time, prepare the master mix for the PCR reaction.		#*In the mean time, prepare the master mix for the PCR reaction.
-	#*The boiled samples are stable at room temp for ~~sometime~~. Keep on ice or freeze for longer.	+	#*The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.

	===PCR===		===PCR===

Tetm2002 — Mon, 12 Jun 2006 04:28:45 GMT

Notes

←Older revision		Revision as of 04:28, 12 June 2006
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	==Notes==		==Notes==
		+	*Note that we use 0.5uL of 100uM primer, which is about ten-times more than standard PCR protocols.
	*The PCR product can be loaded onto agarose gels directly without addition of loading buffer.		*The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
	*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.		*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
-		+	*The expected PCR product should be as short as possible. Anything less than 1kbp can be easily amplified.
-		+

	==Contact==		==Contact==
	[[Special:Emailuser/Tetm2002\|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])		[[Special:Emailuser/Tetm2002\|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])

Tetm2002 — Mon, 12 Jun 2006 04:23:11 GMT

Contact

←Older revision		Revision as of 04:23, 12 June 2006
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	==Contact==		==Contact==
-	[[Special:Emailuser/Tetm2002\|Tet]]	+	[[Special:Emailuser/Tetm2002\|Tet]] ([http://biochemistry.ucsf.edu/~blackburn/ Blackburn Lab])

Tetm2002 — Mon, 12 Jun 2006 04:22:39 GMT

Contact

←Older revision		Revision as of 04:22, 12 June 2006
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	==Contact==		==Contact==
-	*Who has experience with this protocol?	+	[[Special:Emailuser/Tetm2002\|Tet]]

Tetm2002 — Mon, 12 Jun 2006 04:21:57 GMT

References

←Older revision		Revision as of 04:21, 12 June 2006
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	*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.		*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.

-	~~==References==~~	+
-	~~'''Relevant papers and books'''~~	+
-	~~<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->~~	+
-	~~<biblio>~~	+
-	~~#Goldbeter-PNAS-1981 pmid=6947258~~	+
-	~~#Jacob-JMB-1961 pmid=13718526~~	+
-	~~#Ptashne-Genetic-Switch isbn=0879697164~~	+
-	~~</biblio>~~	+

	==Contact==		==Contact==
	*Who has experience with this protocol?		*Who has experience with this protocol?

Tetm2002 — Mon, 12 Jun 2006 04:21:45 GMT

Notes

←Older revision		Revision as of 04:21, 12 June 2006
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	==Notes==		==Notes==
-	~~#List troubleshooting tips here.~~	+	*The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
-	~~#You~~ can ~~also link to FAQs/tips provided by other sources such as the manufacturer or other websites.~~	+	*For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.
-	~~#Anecdotal observations that might~~ be of ~~use to others can also be posted here~~.	+
-		+
-	~~Please sign your name to your note by adding <font face="courier"><nowiki>('''~~~~''')</nowiki></font> to~~ the ~~end~~ of ~~your tip~~.	+

	==References==		==References==

Tetm2002 — Mon, 12 Jun 2006 04:19:41 GMT

Procedure

←Older revision		Revision as of 04:19, 12 June 2006
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	==Procedure==		==Procedure==
-	#~~Step 1~~	+
-	#~~Step 2~~	+	===Yeast Cell Lysis===
-	#*~~Step 2 has some additional information that goes with it~~. i.~~e. Keep~~ at ~~4°C~~.	+	#Aliquot 3uL of 0.02M NaOH into PCR tubes.
-	#~~Step 3~~	+	#Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
-	##~~Step 3 has multiple sub~~-~~steps within it.~~	+	#*If the solution is cloudy, you've added enough cells.
-	##~~Enumerate~~ each of ~~those~~.	+	#[optional] Quick-freeze the cells in liquid nitrogen
		+	#*I normally skip this step. Works just fine.
		+	#Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
		+	#*In the mean time, prepare the master mix for the PCR reaction.
		+	#*The boiled samples are stable at room temp for sometime. Keep on ice or freeze for longer.
		+
		+	===PCR===
		+
		+	#Prepare the master mix solution containing:
		+	#*5uL 5X Q-solution
		+	#*2.5uL 10X PCR Buffer
		+	#*0.5uL dNTPs (10mM each)
		+	#*0.5uL foward primer (100uM)
		+	#*0.5uL reverse primer (100uM)
		+	#*0.25uL Taq
		+	#*12.75uL ddH2O
		+	#Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
		+	#Run the following PCR cycle:
		+	##5 min at 94C
		+	##30 cycles of:
		+	###30 sec at 94C
		+	###30 sec at 55C (or appropriate annealing temperature)
		+	###1 min/kbp at 72C
		+	##10 min at 72C

	==Notes==		==Notes==

Tetm2002 — Mon, 12 Jun 2006 04:01:46 GMT

Materials

←Older revision		Revision as of 04:01, 12 June 2006
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	==Materials==		==Materials==

-	Standard PCR machine, tubesQiagen Taq with Q-solution	+	*Standard PCR machine, tubes
	*[http://www.qiagen.com/Products/Pcr/TaqSystem/TaqDnaPolymerase.aspx Qiagen Taq Polymerase Kit with Q-solution]		*[http://www.qiagen.com/Products/Pcr/TaqSystem/TaqDnaPolymerase.aspx Qiagen Taq Polymerase Kit with Q-solution]
	*A small yeast colony		*A small yeast colony